The skin produces urocortin

Since the skin produces POMC peptides, in the present work we investigated local production of urocortin, a peptide related to CRH, the normal endogenous stimulant for POMC. Urocortin immunoreactivity was detected by direct RIA in extracts of human skin, mouse skin (C57BL-6 strain), cultured cells from established lines of human melanoma and squamous cell carcinoma, human keratinocytes (Ha-CaT), and hamster melanomas. Addition of a reverse phase high performance liquid chromatography step before the RIA confirmed the presence of urocortin, as the immunoreactivity eluted at the same retention time as urocortin standard in extracts from HaCaT keratinocytes and mouse skin. Using the tandem technique of liquid chromatography-mass spectrometry, we identified a peptide with the same mass and retention time as the urocortin standard in human skin extracts. The urocortin antigen could be immunolocalized to normal keratinocytes of the epidermis and hair follicle, epithelium of sweat and sebaceous glands, dermal skeletal muscle, and nevocytes; it was also detected in melanoma and basal cell carcinoma cells. RT-PCR amplification of ribonucleic acid from human skin, cultured keratinocytes, and melanoma cells showed a 145-kb fragment from the coding region of exon 2 of the urocortin gene in all of the tested sources. Lastly, sequencing of the amplified fragment confirmed 100% homology with the known sequence of the urocortin gene. In conclusion, we now demonstrate that human skin and mouse skin as well as cultured keratinocytes and melanoma cells exhibit functional expression of the urocortin gene with actual production of urocortin peptide.